PROJECT SUMMARY/ABSTRACT: P1. Interrogating Distinct Tumor-Initiating Cells A major therapeutic barrier in advanced colorectal cancer (CRC) is tumor recurrence after treatment. The ?cancer stem cell hypothesis? posits that rare populations of cancer cells with stem cell characteristics, also known as tumor-initiating cells (TICs), fuel tumor growth, resist therapy, and repopulate the tumor at distal sites. In the normal colon, multiple stem cell populations exist in a reserve-to-active continuum. We hypothesize that distinct TIC populations exist in CRC, and these distinct populations of TICs have different clonogenic properties and tumor-repopulating potential. Using TICs marked by Lrig1 and Lgr5 to represent reserve and active TICs, respectively, we will use novel single-cell approaches to investigate the behaviors of these populations in the context of therapeutic intervention. First, we will determine whether there is a defined directional hierarchy where reserve TICs give rise to active TICs versus mutual interconversion. Second, we will determine whether TICs with malignant characteristics pre-exist in the primary tumor or if TICs acquire these characteristics de novo as a result of treatment. Third, we will determine whether a signature derived from TIC behaviors provides prognostic and/or predictive information for individuals with CRC. Our translational goal is to apply a systems approach to predict likelihood of tumor recurrence based on properties of TIC populations with the long-term goal of using combinatorial pathway alterations to target TIC behaviors.